Saccharide-measuring fluorescent monomer, saccharide-measuring fluorescent sensor substance, and implantable, saccharide-measuring sensor

ABSTRACT

A fluorescent monomer compound represented by the following formula (1) is provided: 
                         
wherein Q, Q′ and D 3  may be the same or different, may be combined together into a fused ring, and are each a substituent selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, and substituted or unsubstituted alkyl, acyl, oxyalkyl, carboxyl, carboxylate ester, carboxamido, cyano, nitro, amino and aminoalkyl groups; and D 1 , D 2  and D 4  each represent a substituent, wherein at least one of D 1 , D 2  and D 4  is a substituent group comprising a vinyl group at an end thereof, and wherein the substituent group comprising a vinyl group at an end thereof enables the fluorescent monomer compound to be soluble in water.

BACKGROUND OF THE INVENTION

This invention relates to fluorescent monomer compounds, fluorescencesensor substances, their production process, and implantable,saccharide-measuring sensors making use of them. These fluorescentmonomer compounds, fluorescence sensor substances and implantable,saccharide-measuring sensors are excellent in the ability to detectsaccharides.

DESCRIPTION OF THE RELATED ART

Implantable sensors are useful for the progress observation of morbidconditions, the monitoring of therapeutic effects, and the like purposesin various diseases, and their developments have become one of activeresearch fields in recent years. Especially in the treatment ofdiabetes, the control of blood sugar by continuous blood-sugarmeasurement is considered to contribute to a retardation in the progressof morbid conditions and a reduction in the development ofcomplications.

For the self-control of blood sugar, many of current diabetics collectblood samples by punctures of their fingers or the like, and feed themto blood sugar meters to read measurement data. However, this methodinvolves problems of causing pain to patients and in simplicity andeasiness, so that it may not be practical to perform more than severalmeasurements in a day. Under the current circumstances, it is hencedifficult to ascertain the trend of variations in blood sugar levelthrough frequent measurements. For these reasons, implantable,continuous blood sugar meters are considered to have high utility.

On the other hand, technologies have been developed over many years forthe continuous measurement of the in vivo glucose level. Suchtechnologies include, for example, the measurement of a glucose level,which relies upon a change in fluorescence intensity by using asubstance that reversibly reacts with glucose to emit fluorescence. Assuch a fluorescent substance, JP 8-53467 A discloses afluorescence-emitting compound having a molecular structure thatcontains at least one phenylboronic acid moiety and at least oneamine-providing nitrogen atom where the nitrogen atom is disposed in thevicinity of the phenylboronic acid moiety so as to interactintramolecularly with the boronic acid moiety. As the fluorophore, anaphtyl group, anthryl group or the like is used. Upon formation of astable complex with a saccharide molecule via the phenylboronic acidmoiety, the compound emits fluorescence.

As an indicator macromolecule for detecting the concentration of ananalyte in an aqueous environment, WO 02/12251 A1 discloses a copolymerof a hydrophilic monomer and an excimer-forming polycyclic aromatichydrocarbon such as an anthracene derivative. As the excimer-formingpolycyclic aromatic hydrocarbon is not sufficient in water solubility,hydrophilic groups such as methacrylamide groups are introduced suchthat the concentration of an analyte can be detected even in an aqueousenvironment.

Further, U.S. Pat. No. 6,319,540 A1 discloses a process for directlyimmobilizing a fluorescent substance in a solid phase such as a plasticfilm to provide a fluorescence sensor. Employed in U.S. Pat. No.6,319,540 A1 is a fluorescent substance formed of an atomic group, whichhas light emitting property, fluorescence emitting property and colorproducing property, only one phenylboronic acid moiety added to theatomic group.

However, the compound disclosed in JP 8-53467 A contains as afluorophore a bulky hydrophobic moiety such as a naphthyl group oranthryl group, and therefore, its binding to a water-soluble saccharideis not easy. There is, accordingly, an outstanding desire forimprovements in detection sensitivity. Further, the compound disclosedin WO 02/12251 A1 is used as a solution in ethylene glycol uponconducting polymerization with a hydrophilic monomer such as methacrylicacid. The use of an organic solvent upon polymerization, however, has apotential problem in that a gel of such undesired properties asdeveloping variations upon measurement in an aqueous solution may beobtained.

On the other hand, the direct immobilization of a fluorescent substanceon a support material to use the fluorescent substance as a fluorescencesensor may result in the production of smaller signals because there isa limitation to the degree of freedom of the fluorescent substanceimmobilized on the support material. Further, immobilization of thefluorescence substance at a high density may result in quenching. Theability of the fluorescent substance to detect a target substance may,therefore, be reduced compared with that of the same fluorescentsubstance before its immobilization.

SUMMARY OF THE INVENTION

The present invention can provide a fluorescent monomer compoundexcellent in the ability to detect a saccharide such as glucose, afluorescence sensor substance, and a saccharide-measuring sensor makinguse of the fluorescence sensor substance.

The present inventors studied in detail the states of binding ofsaccharides to saccharide-measuring fluorescent monomer compounds. As aresult, it was found that the introduction of one or more hydrophilicgroups, or the introduction of only one hydrophilic group, such as onecomprising a polyalkylene or the like, to a hydrophobic moiety whichemits fluorescence upon binding with a saccharide, makes it possible topromote the binding with the saccharide while maintaining the degree offreedom of the hydrophobic moiety and also that the copolymerization ofthe fluorescent monomer compound with (meth)acrylamide makes it possibleto perform the measurement of a saccharide without a reduction indetection sensitivity even in an aqueous solution such as blood or abody fluid even when the resulting copolymer is immobilized on a supportmaterial. Based on these findings, the present invention has beencompleted.

Saccharide-measuring fluorescent monomer compound, fluorescence sensorsubstance and detector layers according to the present invention areexcellent in the ability to detect saccharides. Owing to their excellentability to detect saccharides in body fluids, these fluorescent monomercompound, fluorescence sensor substance and detector layers can providefluorescence sensors which can withstand their long-term implantation.

The above and other objects, features and advantages of the presentinvention will become clear from the following description of preferredembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing one example of a synthesis scheme for9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid-1-(6-acrylamido-n-hexyl)amide (F-AAm).

FIG. 2 is a diagram showing one example of a synthesis scheme for9,10-bis(methylene)[[N-(ortho-boronobenzyl)methylene]-N-[(acryloylpolyoxyethylene)carbonylamino]-n-hexamethylene]-2-acetylanthracene(F-PEG-AAm-1).

FIG. 3 is a diagram showing one example of a synthesis scheme for methyl9,10-bis(methylene)[[N-(ortho-boronobenzyl)methylene]-N-[(acryloylpolyoxyethylene)carbonylamino]-n-hexamethylene]anthracene-2-carboxylate(F-PEG-AAm-2).

FIG. 4 is a schematic diagram showing one example of a detector layerwith a fluorescence sensor substance according to the present inventionimmobilized on a support material.

FIG. 5 is a perspective view illustrating an external view of animplantable, saccharide-measuring sensor according to the presentinvention.

FIG. 6 is a perspective view depicting the internal structure of theimplantable, saccharide-measuring sensor.

FIG. 7 is a chart showing glucose responses of fluorescence sensorsubstances of Examples 3-10.

FIG. 8 is a chart showing glucose responses of fluorescence sensorsubstances of Examples 3-9.

FIG. 9 is a chart showing glucose responses of detector layers ofExample 16 and Comparative Example 1.

FIG. 10 is a chart showing glucose responses of detector layers ofExample 18, Example 20 and Comparative Example 2.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In a first aspect of the present invention, there is provided afluorescent monomer compound represented by the following formula (1):

wherein: Q, Q′ and D³ may be the same or different, may be combinedtogether into a fused ring, and are each a substituent selected from thegroup consisting of a hydrogen atom, a halogen atom, a hydroxyl group,and substituted or unsubstituted alkyl, acyl, oxyalkyl, carboxyl,carboxylate ester, carboxamido, cyano, nitro, amino and aminoalkylgroups, and preferably, Q and Q′ may each be a hydrogen atom or anacetyl or nitro group; and at least one substituent selected from thegroup consisting of D¹, D² and D⁴ is a substituent group which comprisesa vinyl group at an end thereof, and wherein the substituent groupcomprising a vinyl group at an end thereof enables the fluorescentmonomer compound to be soluble in water. The inclusion of a vinyl groupat the end facilitates the polymerization of the fluorescent monomercompound itself, the polymerization with another polymerizable monomer,and the immobilization on the support material.

The expression “enables the fluorescent monomer compound to be solublein water” as used herein means that the fluorescent monomer compound canbe dissolved at a concentration of 1 mM or higher in water underconditions of 25° C. temperature and pH 7.0 without the presence of anyorganic solvent or solubilizer. As the substituent group that can enablethe fluorescent monomer compound to be soluble in water, a substituentgroup represented by the below-described formula (2) or formula (3) canbe mentioned preferably. Details of the substituent groups representedby the formula (2) or formula (3) will be described subsequently herein.More preferably, D¹, D 2, D³ and D⁴ can have meanings under thefollowing definitions (i) or (ii):

(i) D¹ and D² may be the same or different and are each a substituted orunsubstituted alkyl group, D³ is a hydrogen atom, and D⁴ is asubstituent group represented by the below-described formula (2). As thealkyl group, methyl, ethyl, propyl, butyl, pentyl or the like ispreferred, with methyl or ethyl being more preferred.

(ii) D¹ and D² may be the same or different and are each a substituentgroup represented by the following formula (3), and D³ and D⁴ may be thesame or different, may be combined together into a fused ring, and areeach a substituent selected from the group consisting of a hydrogenatom, a halogen atom, a hydroxyl group, and substituted or unsubstitutedalkyl, acyl, oxyalkyl, carboxyl, carboxylate ester, carboxamido, cyano,nitro, amino and aminoalkyl groups. As the alkyl groups, those having 1to 10 carbon atoms are preferred. Specific examples include methyl,ethyl, propyl, butyl and pentyl. Examples of the acyl groups includeformyl, acetyl, propionyl, butyryl and isobutyryl. Examples of theoxyalkyl groups include methoxy and ethoxy. Examples of the halogenatoms include F, Cl, Br and I. Examples of the aminoalkyl groups includemethylamino and ethylamino. Introduction of nitro, cyano and/or acylgroups as Q, Q′, D³ and D⁴ can bring about an effect that contributes tothe red-shift of fluorescence or the widening of the distance between anexcitation wavelength peak and a fluorescence wavelength peak, therebyfacilitating an analysis of the results of fluorometry.

When D¹, D², D³ and D⁴ have the meanings of the definition (ii) in thepresent invention, it is preferred that at least one of Q, Q′, D³ and D⁴is a substituent group selected from acetyl, carboxylate ester and cyanogroups and the remainder(s) is(are) hydrogen atom(s).

A description will first be made of a case in which D¹, D², D³ and D⁴have the meanings of the definition (i) in the formula (1).

In the definition (i), X is a substituent group selected from the groupconsisting of —COO—, —OCO—, —CH₂NR—, —CH₂S—, —CH₂O—, —NR—, —NRCO—,—CONR—, —SO₂NR—, —NRSO₂—, —O—, —S—, —SS—, —NRCOO—, —OCONR— and —CO—.Examples of the alkyl group include methyl, ethyl, propyl, butyl andpentyl. Preferred examples of X include —NRCO— and —CONR—.

In the definition (i), R″ represents a hydrogen atom or a substituted orunsubstituted alkyl group. As the alkyl group, one having 1 to 10 carbonatoms is preferred, with one having 1 to 5 carbon atoms being morepreferred. Specific examples include methyl, ethyl, propyl, butyl andpentyl.

In the definition (i), Y is a substituted or unsubstituted, divalent,organic residual group, and enables the fluorescent monomer compound tobe soluble in water. The expression “enables the fluorescent monomercompound to be soluble in water” as used herein means that thefluorescent monomer compound can be dissolved at a concentration of 1 mMor higher in water under conditions of 25° C. temperature and pH 7.0without the presence of any organic solvent or solubilizer. Preferredexamples of Y include those containing one or more hydrophilic groupssuch as amino, carboxy, sulfo, nitro, amino, phosphate and/or hydroxylgroups; and those containing one or more hydrophilic linkages such asether, amide and/or ester linkages in their structures.

Further, Y may preferably contain, in the organic residual group, astructure represented by the below-described formula (4) or (5). Y mayadditionally contain one or more other substituent groups and/ordivalent, organic residual groups. In the formula (4) and formula (5), ncan preferably be from 2 to 4, with 2 to 3 being more preferred; j canpreferably be from 1 to 3, with 1 being more preferred; and m canpreferably be from 20 to 150, with 40 to 120 being more preferred. Y′and Y″ may be the same or different, and are each a hydrogen atom oralkyl group. As the alkyl group, one having 1 to 4 carbon atoms ispreferred. Illustrative are methyl, ethyl, propyl and butyl. As Y′ andY″, it is particularly preferred that Y′ and Y″ are each a hydrogen atomor that Y′ is a hydrogen atom and Y″ is an alkyl group having 1 to 4carbon atoms, notably a methyl group.

Y can preferably have number of atoms of from 3 to 500, with 3 to 12being more preferred.

A description will next be made of a case in which D¹, D², D³ and D⁴have the meanings of the definition (ii) in the formula (1).

In the definition (ii), X represents a C₁-C₃₀ alkylene group containingat least one substituent group selected from the group consisting of—COO—, —OCO—, —CH₂NR—, —NR—, —NRCO—, —CONR—, —SO₂NR—, —NRSO₂—, —O—, —S—,—SS—, —NRCOO—, —OCONR— and —CO—. R represents a hydrogen atom or asubstituted or unsubstituted alkyl group. The expression “alkylene groupcontaining at last one substituent group” as used herein means analkylene group containing at least one substituent group at an endthereof or an alkylene group containing at least one substituent groupin the chain thereof. The carbon number of the alkylene group may bepreferably from 1 to 30, more preferably from 3 to 12. Specific examplesinclude propylene, hexylene and octylene. As the at least onesubstituent group contained in the alkylene group, —NRCO— or —CONR— ispreferred. When R is an alkyl group, one having 1 to 10 carbon atomsbeing preferred, with 1 to 5 being more preferred. Specific examplesinclude methyl, ethyl, propyl, butyl and pentyl. As R, a hydrogen atomis preferred.

In the definition (ii), Z represents —O— or —NR″—, and R″ represents ahydrogen atom or a substituted or unsubstituted alkyl group. As thealkyl group, one having 1 to 10 carbon atoms is preferred, with onehaving 1 to 5 carbon atoms being more preferred. Specific examplesinclude methyl, ethyl, propyl, butyl and pentyl. As Z, —O— is preferred.

In the definition (ii), Y is a substituted or unsubstituted, divalent,organic residual group, and enables the fluorescent monomer compound tobe soluble in water. The expression “enables the fluorescent monomercompound to be soluble in water” as used herein means that thefluorescent monomer compound can be dissolved at a concentration of 1 mMor higher in water under conditions of 25° C. temperature and pH 7.0without the presence of any organic solvent or solubilizer. Preferredexamples of Y include those containing one or more hydrophilic groupssuch as amino, carboxy, sulfo, nitro, amino, phosphate and/or hydroxylgroups; and those containing one or more hydrophilic linkages such asether, amide and/or ester linkages in their structures.

In the definition (ii), Y can preferably have a molecular weight of from500 to 10,000, with 1,000 to 5,000 being more preferred.

Owing to the introduction of the hydrophilic chains Y as acharacteristic feature of the present invention, the present inventioncan bring about advantageous effects such as, for example, those to bedescribed below under (1), (2), (3) and (4).

(1) As the fluorescent monomer compound is enabled to be soluble inwater, it is possible to efficiently conduct immobilization and apolymerization reaction upon forming a fluorescence sensor substance.Upon preparation of an acylamide gel, for example, polymerization isfeasible using only water as a solvent, and one having high physicalstrength, stability and uniformity can be obtained. With a hydrophobicmonomer compound, use of an organic solvent or the like is needed forits solubilization, and a gel of undesired properties may be obtained.(2) The introduction of the hydrophilic chains can modify theenvironment and mobility around phenylboronic acid moieties whichinteract with an analyte, thereby contributing to improvements insensitivity, accuracy, response speed, and the selectivity to asaccharide as the analyte. (3) The hydrophilic chains can stabilize thefluorescence sensor substance in its entirety, for example, itspolymerized structure. (4) As the fluorescent monomer compound can bereacted in water alone, its polymerization is feasible even on a supportmaterial susceptible to attacks by an organic solvent, for example, onan plate made of an acrylic resin or the like.

The fluorescent monomer compound according to the present invention ischaracterized in that Y is introduced via X into a compound fordetecting a saccharide. This has made it possible to provide thefluorescent monomer compound with improved physical properties,stability, detection sensitivity, detection accuracy, and selectivity tothe saccharide as the analyte. Described specifically, the introductionof hydrophilic groups represented by the formula (4) or formula (5)leads to an improvement in the degree of freedom at the phenylboronicacid moieties, which are contained in the fluorescent monomer compound,in an aqueous solution such as blood or a body fluid, thereby making itpossible to promptly interact with a saccharide. As a consequence, theaffinity to the saccharide can be increased to improve the detectionsensitivity.

Especially in the case of the definition (i) in the formula (1), onlyone hydrophilic group has been introduced in a hydrophobic moiety whichbinds to a saccharide to emit fluorescence. Accordingly, the bindingwith the saccharide can be promoted while retaining a degree of freedomat the hydrophobic moiety.

It is known that, as described above, the fluorescent monomer compoundaccording to the present invention is a phenylboronic acid derivativewith an anthracene skeleton included therein and the anthracene skeletonacts as a fluorophore. When the phenylboronic acid moieties and asaccharide form stable complexes, fluorescence is emitted owing to theinclusion of the fluorophore. As the fluorescent monomer compoundaccording to the present invention contains two phenylboronic acidmoieties, it is excellent especially in the detection sensitivity tosaccharides. It is to be noted that COCHCH₂ bonded with Z in the formula(3) has been introduced to bind the fluorescent monomer compound to asupport material or the like such that the fluorescent monomer compoundis prevented from dissolution in a body flood such as blood.

In the second aspect of the present invention, there is provided asaccharide-measuring fluorescence sensor substance comprising acopolymer of at least the following two compounds (I) and (II):

-   (I) a fluorescent monomer compound represented by the formula (1),    and-   (II) at least one hydrophilic, non-fluorescent, polymerizable    monomer having a vinyl group.

To detect a saccharide, which is contained in an aqueous solution suchas a blood or a body fluid, by using the fluorescent monomer compound,immobilization of the fluorescent monomer compound is needed to preventthe fluorescent monomer compound from being dissolved in or flowing outinto the aqueous solution. When the fluorescent monomer compound issimply immobilized on a support material, however, the contact andbinding between the fluorescent monomer compound and the saccharide isinhibited, leading to a reduction in detection sensitivity. In thepresent invention, the fluorescent monomer compound and the at least onehydrophilic, non-fluorescent, polymerizable monomer containing the vinylgroup are, therefore, copolymerized to introduce and immobilize thehydrophilic polymerizable monomer in the fluorescent monomer compound sothat the fluorescence sensor substance is formed. This has made itpossible to make the fluorescent monomer compound insoluble whileassuring high affinity between the fluorescent monomer compound and thesaccharide.

Upon preparation of the fluorescence sensor substance, theabove-mentioned at least one hydrophilic, non-fluorescent, polymerizablemonomer having the vinyl group can be dissolved in water even at aconcentration required for polymerization. When the fluorescence sensorsubstance is prepared in the form of a gel, for example, the gel canhence be obtained with such desired properties as scarcely developingvariations in measurement data. Upon preparing the fluorescence sensorsubstance with the at least one hydrophilic, non-fluorescent,polymerizable monomer having the vinyl group, the concentration of themonomer in a reaction mixture can range preferably from 0.5 to 50 wt %,more preferably from 3 to 30 wt %.

As the at least one hydrophilic, non-fluorescent, polymerizable monomerhaving the vinyl group, a polymerizable monomer having an acrylic acidresidual group or a polymerizable monomer having a (meth)acrylamideresidual group can be preferably mentioned, with the polymerizablemonomer having the (meth)acrylamide residual group being more preferred.The polymerizable monomer having the (meth)acrylamide residual group isexcellent especially in water solubility and operation ease.

No particular limitation is imposed on the polymerizable monomer havingan acrylic acid residual group insofar as the resulting polymer containsacryloyl groups in its structure. Examples can include 4-hydroxydibutylacrylate, 2-hydroxyethyl acrylate, hydroxypropyl acrylate, methoxyethylacrylate, polyethyleneglycol acrylate, and acrylic acid.

No particular limitation is imposed on the polymerizable monomer havingthe (meth)acrylamide residual group insofar as the resulting polymercontains acryloyl groups and amide linkages in its structure. Examplescan include (meth)acrylamide and its derivatives. Illustrative arecondensation products between (meth)acryloyl chloride and amino acids orcompounds containing active amino groups, such as acrylamide,methacrylamide, N,N-dimethylacrylamide, N-tris-hydroxymethylacrylamide,N-hydroxymethylacrylamide, N-(n-butoxymethyl)acrylamide, N-acryloyllysine, and N-acryloylhexamethylenediamine; and compounds represented bythe following formula (6):

In the formula (6), A is a hydrogen atom or a methyl group, and U and U′may be the same or different and are each a substituted or unsubstitutedalkyl group. Examples of the alkyl group include methyl, ethyl, propyl,butyl, and pentyl.

The polymer, which comprises the polymerizable monomer with the(meth)acrylamide residual group contained therein, has highhydrophilicity so that, when bonded with the fluorescent monomercompound, the fluorophores which exist in the fluorescent monomercompound, contain the phenylboronic acid moieties and have stronghydrophobicity are incorporated in the highly hydrophilic structure.Even when a saccharide contained in blood or a body fluid is to bemeasured, water-soluble saccharide can, therefore, easily approach andbind to the fluorophores.

The molar ratio [(I):(II)] of the fluorescent monomer compound (I) tothe polymerizable monomer containing the acrylamide residual group inthe copolymer may be preferably from 1:10 to 1:4,000, more preferablyfrom 1:50 to 1:4,000, especially preferably from 1:100 to 1:2,000 whenD¹, D², D³ and D⁴ have the meanings of the definition (i) in thefluorescent monomer compound. A proportion of the fluorescent monomercompound (I) greater than that giving the molar ratio of 1:10 involves apotential problem that the degree of freedom may be lost due to thebulkiness of the hydrophobic moieties of the fluorescent monomercompound and the interaction with a saccharide may be reduced. Aproportion of the fluorescent monomer compound (I) smaller than thatgiving the molar ratio of 1:4,000, on the other hand, may not be able toassure the absolutely-needed level of fluorescence intensity.

The molar ratio [(I):(II)] of the fluorescent monomer compound (I) tothe polymerizable monomer containing the acrylamide residual group inthe copolymer may be preferably from 1:50 to 1:6,000, more preferablyfrom 1:150 to 1:3,000 when D¹, D², D³ and D⁴ have the meanings of thedefinition (ii) in the fluorescent monomer compound. A proportion of thefluorescent monomer compound (I) greater than that giving the molarratio of 1:50 involves a potential problem that the degree of freedommay be lost due to the bulkiness of the hydrophobic moieties in thefluorescent monomer compound and the interaction with a saccharide maybe reduced. A proportion of the fluorescent monomer compound (I) smallerthan that giving the molar ratio of 1:6,000, on the other hand, may notbe able to assure the absolutely-needed level of fluorescence intensity.

The weight average molecular weight of the fluoresence sensor substancecomposed of the above-described two components can be preferably from50,000 to 500,000, more preferably from 100,000 to 300,000, asdetermined by GPC using a polyethylene oxide standard, when D¹, D², D³and D⁴ have the meanings of the definition (i) in the fluorescentmonomer compound.

The weight average molecular weight of the fluoresence sensor substancecan be preferably from 50,000 to 750,000, more preferably from 150,000to 450,000, as determined by GPC using a polyethylene oxide standard,when D¹, D², D³ and D⁴ have the meanings of the definition (ii) in thefluorescent monomer compound.

The fluorescence sensor substance according to the present invention mayuse one or more other components in addition to the fluorescent monomercompound and the poymerizable monomer containing the (meth)acrylamideresidual group. Examples of such components include crosslinkablemonomers, other crosslinkable components, cationic monomers cable ofproviding cations in water, anionic monomers capable of providing anionsin water, and nonionic monomers containing no groups capable ofliberating ions in water.

Examples of the crosslinkable monomers include a wide variety of thosecapable of introducing a three-dimensional crosslinked structure intothe fluorescence sensor substance via polymerizable double bonds.Illustrative are divinyl compounds such as N,N′-methylenebis(meth)acrylamide, N,N′-(1,2-dihydroxyethylene)-bis(meth)acrylamide,diethylene glycol di(meth)acrylate, (poly)ethylene glycoldi(meth)acrylate, triethylene glycol di(meth)acrylate, propylene glycoldi(meth)acrylate, trimethylolpropane di(meth)acrylate,trimethylolpropane tri(meth)acrylate, pentaerythritol di(meth)acrylate,pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate,(poly)propylene glycol di(meth)acrylate, glycerin tri(meth)acrylate,glycerin acrylate methacrylate, ethylene-oxide-modifiedtrimethylolpropane tri(meth)acrylate, pentaerythritoltetra(meth)acrylate, and dipentaerythritol hexa(meth)acrylate, althoughthey differ depending on the substituent groups in the fluorescencesensor substance to be used. Two or more of these crosslinkable monomerscan be used in combination in the present invention.

Examples of the other crosslinkable components include a wide variety ofcompounds each containing two or more functional groups. Illustrativeare triallyl cyanurate, triallyl isocyanurate, triallyl phosphate,triallylamine, poly(meth)allyloxyalkanes, (poly)ethylene glycoldiglycidyl ether, glycerol diglycidyl ether, ethylene glycol,polyethylene glycol, propylene glycol, glycerin, pentaerythritol,ethylenediamine, polyethyleneimine, glycidyl (meth)acrylate, triallylisocyanurate, trimethylol propane di(meth)allyl ether,tetraallyloxyethane, and glycerol propoxytriacrylate, although theydiffer depending on the substituent groups in the fluorescence sensorsubstance to be used. Two or more of these crosslinkable monomers can beused in combination in the present invention.

Examples of the cationic monomers cable of providing cations in waterinclude dimethylaminoethyl (meth)acrylate, diethylaminoethyl(meth)acrylate, and 4-vinylpyridine. Two or more of these cationicmonomers can be used in combination in the present invention.

Examples of the anionic monomers capable of providing anions in waterinclude (meth)acrylic acid, vinylpropionic acid, and4-vinylbenzenesulfonic acid. Two or more of these anionic monomers canbe used in combination in the present invention.

Examples of the nonionic monomers containing no groups capable ofliberating ions in water include 2-hydroxyethyl (meth)acrylate,3-methoxypropyl (meth)acrylate, 4-hydroxydibutyl (meth)acrylate,2-methoxyethyl acrylate, and 1,4-cyclohexanedimethanol monoacrylate. Twoor more of these nonionic monomers can be used in combination in thepresent invention.

Further, two or more of these crosslinkable monomers, othercrosslinkable components, cationic monomers, anionic monomers andnonionic monomers can also be used in combination. The proportion of oneor more of these other components can be preferably from 0.1 to 10 mole%, more preferably from 2 to 7 mole % based on the total proportion ofthe fluorescent monomer compound and the polymerizable monomer with the(meth)acrylamide residual group contained therein. The combined use ofone or more of these other components makes it possible to form athree-dimensional crosslinked structure, and also to effect anadjustment of hydrophilicity, the introduction of starting points for areaction, and the like. Concerning three-dimensional crosslinkedstructures, a description will be made subsequently herein.

The fluorescence sensor substance according to the present invention canpreferably have a structure represented by the following formula (7):

In the formula (7), Q and Q′ are as defined in connection with thefluorescent monomer compound represented by the formula (1). D¹⁰, D²⁰,D³⁰ and D⁴⁰ have meanings of the following definition (x) or (xx):

(x) D¹⁰ and D²⁰ may be the same or different and are each a substitutedor unsubstituted alkyl group, D³⁰ is a hydrogen atom, and D⁴⁰ is asubstituent group represented by the below-described formula (8). As thealkyl group, methyl, ethyl, propyl, butyl, pentyl or the like ispreferred, with methyl or ethyl being more preferred.

(xx) D¹⁰ and D²⁰ may be the same or different and are each a substituentgroup represented by the below-described formula (9), and D³⁰ and D⁴⁰may be the same or different, may be combined together into a fusedring, and are each a substituent selected from the group consisting of ahydrogen atom, a halogen atom, a hydroxyl group, and substituted orunsubstituted alkyl, acyl, oxyalkyl, carboxyl, carboxylate ester,carboxamido, cyano, nitro, amino and aminoalkyl groups. As the alkylgroup, one having 1 to 10 carbon atoms is preferred. Specific examplesinclude methyl, ethyl, propyl, butyl, and pentyl. Examples of the acylgroup include formyl, acetyl, propionyl, butyryl, and isobutyryl.Examples of the oxyalkyl group include methoxy and ethoxy. Examples ofthe halogen atoms include F, Cl, Br, and I. Examples of the aminoalkylgroup include methylamino and ethylamino. Introduction of nitro, cyanoand/or acyl groups as Q, Q′, D³⁰ and D⁴⁰ can bring about an effect thatcontributes to the red-shift of fluorescence or the widening of thedistance between an excitation wavelength peak and a fluorescencewavelength peak.

When D¹⁰, D²⁰, D³⁰ and D⁴⁰ have the meanings of the definition (xx) inthe present invention, it is preferred that at least one of Q, Q′, D³⁰and D⁴⁰ is a substituent group selected from acetyl, carboxylate esterand cyano groups and the remainder(s) is(are) hydrogen atom(s).

A description will first be made of a case in which D10, D20, D30 andD40 have the meanings of the definition (x) in the formula (8).

In the definition (x), X is a substituent group selected from the groupconsisting of —COO—, —OCO—, —CH₂NR—, —CH₂S—, —CH₂O—, —NR—, —NRCO—,—CONR—, —SO₂NR—, —NRSO₂—, —O—, —S—, —SS—, —NRCOO—, —OCONR— and —CO—.Examples of the alkyl group include methyl, ethyl, propyl, butyl andpentyl. Preferred examples of X include —NRCO— and —CONR—.

In the definition (x), Y and R″ are as defined in connection with thefluorescent monomer compound represented by the formula (1), and U, U′and A are as defined in connection with the polymerizable monomercontaining the (meth)acrylamide residual group, which is represented bythe formula (6).

In the definition (x), the molar ratio (p:q) of p to q in the formula(8) can be preferably from 1:10 to 1:4,000, more preferably from 1:50 to1:4,000, particularly preferably from 1:100 to 1:2,000. A value of pgreater than that giving the molar ratio of 1:10 involves a potentialproblem that the degree of freedom may be lost due to the bulkiness ofthe hydrophobic moieties and the interaction with a saccharide may bereduced. A value of p smaller than that giving the molar ratio of1:4,000, on the other hand, may not be able to assure theabsolutely-needed level of fluorescence intensity.

A description will next be made of a case in which D¹⁰, D²⁰, D³⁰ and D⁴⁰have the meanings of the definition (xx) in the formula (7).

In the definition (xx), X represents a C1-C30 alkylene group containingat least one substituent group selected from the group consisting of—COO—, —OCO—, —CH₂NR—, —NR—, —NRCO—, —CONR—, —SO₂NR—, —NRSO₂—, —O—, —S—,—SS—, —NRCOO—, —OCONR— and —CO—. R represents a hydrogen atom or asubstituted or unsubstituted alkyl group. The expression “alkylene groupcontaining at least one substituent group” as used herein means analkylene group containing at least one substituent group at an endthereof or an alkylene group containing at least one substituent groupin the chain thereof. The carbon number of the alkylene group may bepreferably from 1 to 30, more preferably from 3 to 12. Specific examplesinclude propylene, hexylene and octylene. As the at least onesubstituent group contained in the alkylene group, —NRCO— or —CONR— ispreferred. When R is an alkyl group, one having 1 to 10 carbon atomsbeing preferred, with 1 to 5 being more preferred. Specific examplesinclude methyl, ethyl, propyl, butyl and pentyl. As R, a hydrogen atomis preferred.

In the definition (xx), Y and Z are as defined in connection with thefluorescent monomer compound represented by the formula (1), and U, U′and A are as defined in connection with the polymerizable monomercontaining the (meth)acrylamide residual group, which is represented bythe formula (5).

In the definition (xx), the molar ratio (p:q) of p to q in the formula(7) can be preferably from 1:50 to 1:6,000, more preferably from 1:150to 1:3,000. A value of p greater than that giving the molar ratio of1:50 involves a potential problem that the degree of freedom may be lostdue to the bulkiness of the hydrophobic moieties and the interactionwith a saccharide may be reduced. A value of p smaller than that givingthe molar ratio of 1:6,000, on the other hand, may not be able to assurethe absolutely-needed level of fluorescence intensity.

In the fluorescence sensor substance useful in the present invention, atleast a portion of the copolymer may form intermolecular crosslinks topresent a three-dimensional crosslinked structure. The formation ofthree-dimensional crosslinks across poly(meth)acrylamide chains ispreferred because the fluorescent monomer compound becomes resistant toflowing out. It is to be noted that, although the fluorescent monomercompound according to the present invention contains hydrophobicmoieties capable of emitting fluorescence upon binding a saccharide asdescribed above, the hydrophobic moieties are assured to retain such adegree of freedom as permitting the binding of the saccharide in anaqueous solution because the hydrophobic moieties are bonded topoly(meth)acrylamide chains via their divalent, organic residual groupsrepresented by Y. The detection sensitivity for the saccharide is,therefore, not lowered despite the formation of the three-dimensionalcrosslinked structure.

Although no particular limitations are imposed on the productionprocesses of the fluorescent monomer compound and fluorescence sensorsubstance according to the present invention and the method of formationof the three-dimensional crosslinked structure, they can be produced bythe following processes.

(1-1) Production process of a fluorescent monomer compound In accordancewith a synthesis scheme shown in FIG. 1, a description will be madeabout one example of a process for the production of a compound of theformula (1) in which Q and Q′ are each a hydrogen atom, D¹ and D² areeach a methyl group, D³ is a hydrogen, D⁴ is a substituent grouprepresented by the formula (2), X is —CONH, Y is —C₆H₁₂— and R″ is ahydrogen atom(9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid-1-(6-acrylamido-n-hexyl)amide).

Using methyl-9,10-dimethylanthracene-2-carboxylic acid as a rawmaterial, N-bromosuccinimide (NBS) is reacted with it to yield methyl9,10-bis(bromomethyl)anthracene-2-carboxylate. Subsequent addition ofmethylamine converts the bromomethyl group into an aminomethyl group.2-(2-Bromomethylphenyl)-1,3-dioxaborinane is reacted with the resultantmethyl 9,10-bis(aminomethyl)anthracene-2-carboxylate to obtain methyl9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylate.On the reaction product, an alkali is then caused to act to inducede-esterification, whereby9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid is obtained. Bonding of 1-acrylamido-6-aminohexane to thecarboxylic acid can afford the target compound.

Described more specifically, a solution ofmethyl-9,10-dimethylanthracene-2-carboxylic acid is prepared preferablyat a concentration of from 1 to 20 g/L, with 10 to 15 g/L being morepreferred. NBS is added to the solution such that the molar ratio of NBSto methyl-9,10-dimethylanthracene-2-carboxylic acid ranges preferablyfrom 2 to 2.5 times, more preferably from 2.1 to 2.2 times. In each ofthe above-described synthesis reactions for the target compound, it ispossible to use a solvent suited for the corresponding reaction product.Examples of such a solvent include chloroform, carbon tetrachloride,n-hexane, acetonitrile, dimethylformamide, and dimethylsulfoxide. Thesesolvents can be used either singly or in combination. To dissolvemethyl-9,10-dimethylanthracene-2-carboxylic acid, for example,chloroform, carbon tetrachloride, acetonitrile or the like can besuitably used. Two or more of these solvents may be used in combination,that is, as a mixed solution. Upon using two or more of these solventsin combination, the proportions of such two or more solvents can be setas desired. The reaction temperature is preferably from 60 to 120° C.,more preferably from 80 to 100° C., and the reaction time is preferablyfrom 0.5 to 6 hours, more preferably from 2 to 4 hours.

With methyl 9,10-bis(bromomethyl)anthracene-2-carboxylate which has beendissolved preferably at a concentration of from 1 to 30 g/L, morepreferably at a concentration of from 2 to 10 g/L in a solvent,methylamine is next mixed in a proportion of preferably from 2 to 30molar times, more preferably from 6 to 20 molar times to react them witheach other. The reaction temperature is preferably from 0 to 60° C.,more preferably from 20 to 30° C., and the reaction time is preferablyfrom 1 to 10 hours, more preferably from 2 to 5 hours.

The reaction between the thus-obtained methyl9,10-bis(aminomethyl)anthracene-2-carboxylate and2-(2-bromomethylphenyl)-1,3-dioxaborinane is conducted by mixing themtogether such that the proportion of2-(2-bromomethylphenyl)-1,3-dioxaborinane becomes preferably 2 to 8times, more preferably 3 to 5 times relative to methyl9,10-bis(aminomethyl)anthracene-2-carboxylate. The concentration ofmethyl 9,10-bis(aminomethyl)anthracene-2-carboxylate is preferably from10 to 200 g/L, more preferably from 50 to 100 g/L. The reactiontemperature is preferably from 0 to 80° C., more preferably from 20 to40° C., and the reaction time is preferably from 1 to 48 hours, morepreferably from 2 to 24 hours.

Methyl9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylateis next hydrolyzed with an alkali. As the alkali, any alkaline agent canbe used such as sodium hydroxide or potassium hydroxide. The reactiontemperature is preferably from 0 to 100° C., more preferably from 20 to60° C., and the reaction time is preferably from 1 to 24 hours, morepreferably from 2 to 6 hours.

With9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid (1 mole),1-acrylamido-6-aminohexane (preferably 1.05 to 3.0 moles,more preferably 1.2 to 1.4 moles) and a condensing agent (preferably 1.0to 3.0 moles, more preferably 1.1 to 2.0 moles) are then mixed. As thecondensing agent, dicyclohexylcarbodiimide,1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide or the like can be used.The reaction temperature is preferably from 0 to 60° C., more preferablyfrom 20 to 30° C., and the reaction time is preferably from 1 to 24hours, more preferably from 2 to 15 hours.

It is to be noted that, when a raw compound having, as the substituentsrepresented by Q and Q′ in the formula (1), groups different from thecorresponding groups in the above-described compound is used as theanthracene skeleton, a corresponding compound can be produced by makingsuitable selections as to the solvent, additives, reaction temperature,reaction time, isolation method, etc. Further, use of a compound with asulfonyl group contained on an anthracene skeleton in place of methylcarboxylate makes it possible to introduce SO₂NH— as X. Furthermore,addition of amino-(C₂H₄O)m-acrylamide in place of1-acrylamido-6-aminohexane makes it possible to introduce —(C₂H₄O)m— asY.

It is also to be noted that9,10-bis[[N-methyl-N-(ortho-borobenzyl)amino]methyl]anthracene-2-carboxylicacid can also be prepared by using anthryldiamine-2-carboxylic acid inplace of anthryldiamine in Example 3 of Japanese Patent laid-open No.Hei 8-53467.

(1-2) Production process of another fluorescent monomer compound Inaccordance with a synthesis scheme shown in FIG. 2, a description willbe made about one example of a process for the production of a compoundof the formula (1) in which Q, Q′ and D³ are each a hydrogen atom, D⁴ is—COCH₃, D¹ and D² are each a substituent group represented by theformula (3), D¹ and D² are the same, X is —C₆H₁₂—NHCO—, Y is a PEGresidual group, and Z is—O—{9,10-bis(methylene)[[N-ortho-boronobenzyl)methylene]-N-[(acryloylpolyoxyethylene)carbonylamino]-n-hexamethylene]-2-acetylanthracene(F-PEG-AAm-1)}.

Using 2-acetyl-9,10-dimethylanthracene as a raw material, it is heatedin a carbon tetrachloride/chloroform solvent, and is reacted withN-bromosuccinimide (NBS) and benzoyl peroxide (BPO) to afford2-acetyl-9,10-bis(bromomethylene)anthracene. It is then reacted withN-(t-butoxycarbonyl)-hexyldiamine in the presence of a base such asdiisopropylethylamine (DIEA) in a solvent such as dimethylformamide(DMF). As a result, the bromomethylene group is converted into a[(t-butoxycarbonylamino)hexylamino]methylene group.2-(2-Bromomehtylphenyl)-1,3-dioxaborinane is caused to act on thereaction product in the presence of a base such as DIEA in a solventsuch as DMF to yield9,10-bis[[N-6′-(t-butoxycarbonylamino)hexyl-N-[2-(5,5-dimethylborinane-2-yl)benzyl]amino]methylene]2-acetylanthracene.An acid such as hydrochloric acid is caused to act on the reactionproduct for its deprotection to obtain9,10-bis(methylene)[[N-(ortho-boronobenzyl)methylene]-N-(aminohexyl)]-2-acetylanthracene.It is then reacted with acryloyl-(polyethyleneglycol)-N-hydroxysuccinimide ester to obtain the target compound.

It is to be noted that, when a raw compound having, as the substituentrepresented by D⁴ in the formula (1), a group different from thecorresponding group in the above-described compound is used as theanthracene skeleton, a corresponding compound with a group other than anacetyl group contained as D⁴ can be produced by making suitableselections as to the solvent, additives, reaction temperature, reactiontime, isolation method, etc.

(1-3) Production process of a further fluorescent monomer compound Inaccordance with a synthesis scheme shown in FIG. 3, a description willbe made about one example of a process for the production of a compoundof the formula (1) in which Q, Q′ and D3 are each a hydrogen atom, D⁴ is—COOCH₃, D¹ and D² are each a substituent group represented by theformula (3), D¹ and D² are the same, X is —C₆H₁₂—NHCO—, Y is a PEGresidual group, and Z is —O—{methyl 9,10-bis(methylene)[[N-ortho-boronobenzyl)methylene]-N-[(acryloylpolyoxyethylene)carbonylamino]-n-hexamethylene]anthracene-2-carboxylate (F-PEG-AAm-2)}.

Using 9,10-dimethyl-2-acetylanthracene as a raw material, it is added todioxane/aqueous solution of sodium perchlorate. Subsequent to stirringunder heat, an acid is added to obtain a precipitate of9,10-dimethylacetone-2-carboxylic acid. The precipitate is thendissolved in hydrochloric acid/methanol solvent, and the resultingsolution is heated under reflux to afford methyl9,10-dimethylanthracene-2-carboxlate. The methyl ester is dissolved incarbon a tetrachloride/chloroform solvent. Subsequent to heating, themethyl ester is reacted with N-bromosuccinimide (NBS) and benzoylperoxide (BPO) to convert the methyl ester into methyl9,10-bis(bromomethylene)anthracene-2-carboxylate. The thus-obtainedmethyl ester is then reacted with N-(t-butoxycarbonyl)-hexyldiamine inthe presence of a base such as diisopropylethylamine (DIEA) in a solventsuch as dimethylformamide (DMF). As a result, the bromomethylene groupis converted into a [(t-butoxycarbonylamino)hexylamino]methylene group.2-(2-Bromomethylphenyl)-1,3-dioxaborinane is caused to act on thereaction product in the presence of a base such as DIEA in a solventsuch as DMF to yield methyl9,10-bis[[N-6′-(t-butoxycarbonylamino)hexyl-N-[2-(5,5-dimethylborinan-2-yl)benzyl]amino]methyl]anthracene-2-carboxylate.When deprotected under the action of an acid such as hydrochloric acid,it provides methyl9,10-bis(methylene)[[N-(orthoboronobenzyl)methylene]-N-(aminohexyl)]anthracene-2-carboxylate.It is then reacted with acryloyl-(polyethyleneglycol)-N-hydroxysuccinimide ester in a basic buffer to afford thetarget compound.

It is to be noted that, when a raw compound having, as the substituentrepresented by D⁴ in the formula (1), a group different from thecorresponding groups in the above-described compounds is used as theanthracene skeleton, a corresponding compound with a group other than amethyl carboxylate group contained as D⁴ can be produced by makingsuitable selections as to the solvent, additives, reaction temperature,reaction time, isolation method, etc. (2) production process of afluorescence sensor substance

The copolymerization between the fluorescent monomer compoundrepresented by the formula (1) and the polymerizable monomer containingthe (meth)acrylamide residual group can be conducted using apolymerization accelerator or polymerization initiator in a solvent.When D¹, D², D³ and D⁴ have meanings of the definition (i) in thechemical formula (1), a solvent may preferably be composed of one ormore solvents selected from the group consisting of dimethyl sulfoxide,dimethyl formamide, ethylene glycol and diethylene glycol solvent. Thesolvent may be used as a mixture with water. In particular, use of amixed solution composed of dimethyl sulfoxide or dimethyl formamide andwater can promote the progress of polymerization. When mixed with water,it is preferred to use one containing dimethyl sulfoxide and/or dimethylformamide at a concentration of from 40 to 80 wt %, with 50 to 70 wt %being more preferred. In this concentration range, the mixed solutioncan promote the progress of polymerization and can obtain the targetfluorescence sensor substance with a high yield. It is to be noted thata solvent concentration lower than 40 wt % may result in the initiationof precipitation of the fluorescent monomer compound before theinitiation of polymerization. When D¹, D², D³ and D⁴ have meanings ofthe definition (ii) in the chemical formula (1), water may be used as asolvent. The introduction of Y, which has hydrophilicity of such anextent as permitting providing the fluorescent monomer compound withsolubility in water, has made it possible to conduct polymerization evenwhen water alone is used as a solvent. It is also possible to use waterin the form of a mixture with one or more of dimethyl sulfoxide,dimethyl formamide, ethylene glycol, diethylene glycol and the like.When an organic solvent is mixed in the present invention, its contentis preferably from 10 to 50 wt %, more preferably from 20 to 30 wt %.

Upon copolymerizing the fluorescent monomer compound represented by theformula (1) and the polymerizable monomer containing the(meth)acrylamide residual group, one or more other components may beadded. When one or more other components are added, its or theirproportion is preferably from 0.1 to 10 mole %, more preferably from 2to 7 mole % based on the total proportion of the fluorescent monomercompound and the polymerizable monomer containing the (meth)acrylamideresidual group. When one or more other components are added, it ispreferred to concurrently add a polymerization initiator andpolymerization promoter upon conducting polymerization.

Examples of the polymerization initiator include persulfates such assodium persulfate, potassium persulfate and ammonium persulfate;hydrogen peroxide; azo compounds such as azobis-2-methylpropionamidinehydrochloride and azoisobutyronitrile; and peroxides such as benzoylperoxide, lauroyl peroxide, cumene hydroperoxide and benzoyl peroxide.These polymerization initiators can be used either singly or incombination. In combination with such a polymerization initiator, it isalso possible to use, as a polymerization promoter, one or more ofreducing agents such as sodium hydrogensulfite, sodium sulfate, Mohr'ssalt, sodium pyrobisulfite, sodium formaldehydesulfoxylate, and ascorbicacid; and amine compounds such as ethylenediamine, sodiumethylenediaminetetraacetate, glycine, andN,N,N′,N′-tetramethylethylenediamine. The polymerization temperature ispreferably from 15 to 75° C., more preferably from 20 to 60° C., whilethe polymerization time is preferably from 1 to 20 hours, morepreferably from 2 to 8 hours. Combined use of a persulfate as apolymerization initiator with N,N,N′,N′-tetramethylethylenediamine as apolymerization promoter is particularly preferred in that thepolymerization can be conducted at room temperature.

The compound represented by the formula (7), on the other hand, can alsobe produced without relying upon the copolymerization of the fluorescentmonomer compound represented by the formula (1) with the polymerizablemonomer containing the (meth)acrylamide residual group. As thefluorescent monomer compound represented by the formula (1) issynthesized through plural steps, the fluorescence sensor substancerepresented by the formula (7) can also be eventually produced even wheninstead of using the fluorescent monomer compound represented by theformula (1) as a raw material, another compound is caused to act on anintermediate product for the fluorescent monomer compound. For example,the fluorescence sensor substance represented by the formula (7) canalso be produced even when9,10-bis[[N-(6′-aminohexyl)-N-(ortho-boronobenzyl)amino]methyl]-2-acetylanthraceneshown in the scheme of FIG. 2 or methyl9,10-bis[[N-(6′-aminohexyl)-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylateshown in the scheme of FIG. 3 and one obtained by introducing carboxylgroups into a polymer of the polymerizable monomer, which contains the(meth)acrylamide residual group, are reacted in the presence of acoupling reagent. As another example, the fluorescence sensor substancerepresented by the formula (7) can also be produced when subsequent tothe advance polymerization of the polymerizable monomer containing the(meth)acrylamide residual group, the resulting polymer is copolymerizedwith the fluorescent monomer compound in the presence of apolymerization initiator and polymerization promoter.

(3) Method of formation of a three-dimensional crosslinked structure Itis preferred that at least a portion of the fluorescence sensorsubstance according to the present invention forms intermolecularcrosslinks and has a three-dimensional crosslinked structure. Asmentioned above, the formation of the three-dimensional crosslinkedstructure across poly(meth)acrylamide chains makes the fluorescentmonomer compound resistant to flowing out, and therefore, makes itpossible to detect a saccharide with ease.

No limitation is imposed on the method for the introduction of such athree-dimensional crosslinked structure. For example, intermolecularcrosslinks can be formed between at least some molecules of thefluorescence sensor substance by causing a crosslinking component to acton the fluorescence sensor substance.

As an alternative, such a three-dimensional crosslinked structure canalso be formed by copolymerizing the fluorescent monomer compoundrepresented by the formula (1) and the polymerizable monomer containingthe (meth)acrylamide residual group as described above.

Upon using the fluorescence sensor substance as an implantable,saccharide-measuring sensor, the fluorescence sensor substance isgenerally on a support material to prevent it from flowing out. Theimmobilization on the support material and the formation ofthree-dimensional crosslinks can be effected at the same time by using,as such a support material, the polymerizable monomer containing the(meth)acrylamide residual group or its polymer and polymerizing it withthe fluorescent monomer compound represented by the formula (1) whileusing a crosslinking component as needed.

Usable examples of the crosslinking component include the crosslinkablemonomers, other crosslinkable components, cationic monomers, anionicmonomers and nonionic monomers described above as other components whichcan be added to the fluorescence sensor substance. The crosslinkablemonomers and other crosslinkable components can be used more preferably.These crosslinking components can be used either singly or incombination in the present invention.

In the third aspect of the present invention, there is also provided adetector layer with the above-described fluorescence sensor substanceimmobilized on a support material. In the fourth aspect of the presentinvention, there is also provided an implantable, saccharide-measuringsensor comprising the above-described fluorescence sensor substance ordetector layer.

The implantable, saccharide-measuring sensor may preferably beimmobilized via covalent bonds or hydrophobic bonds or by electrical orother interactions on an immobilizing material such as a supportmaterial such that the fluorescence sensor substance is prevented fromflowing out. An outline of a saccharide detection method making use ofthe fluorescence sensor substance according to the present inventionwill be described with reference to FIG. 4. FIG. 4 is a schematic viewillustrating one example of a detector layer with the fluorescencesensor substance according to the present invention immobilized on asupport material.

The sensor includes a detector layer 10, in which a fluorescence sensorsubstance 30 is immobilized on a support material 40. The fluorescencesensor substance 30 is a copolymer, which at least includes fluorescentmonomer compound moieties 33 indicated by oval dots and polymerizablemonomer moieties 35 having a (meth)acrylamide residual group andindicated by oval circles. Fluorescence is emitted when a saccharide 70in an aqueous solution interacts with the fluorescent monomer compound33. The detector layer 10 may have an optical isolation layer 20. Lightof 350 to 420 nm in wavelength is irradiated from a light source 50 ontothe detector layer 10, and a change in reflected fluorescence intensityor wavelength is detected by a detector 60. As a result, theconcentration of the saccharide can be determined relying upon thefluorescence intensity. Examples of the support material employed in thedetector layer according to the present invention include a wide varietyof materials, for examples, inorganic materials such as glass and metalsand organic materials such as plastic films. Preferred as the supportmaterial for the detector layer for use in the saccharide-measuringsensor is a material that is excellent in transparency and does notdissolve or flow out even in body fluids. Among glass and plastic films,poly(meth)acrylamide films and poly(meth)acrylate films can bepreferably used in the present invention. The use of apoly(meth)acrylamide as a support material is advantageous in that theimmobilization of the fluorescence sensor substance on the supportmaterial and the formation of three-dimensional crosslinked structurecan be effected at the same time as mentioned above. Crosslinkedstructures available from the use of a crosslinkable polymer are formed,as illustrated in FIG. 4, between polymerizable monomer moieties 35,which contain (meth)acrylamide residual groups, themselves, betweenfluorescent monomer moieties 33 and polymerizable monomer moieties 35containing (meth)acrylamide residual groups, and between polymerizablemonomer moieties 35 containing (meth)acrylamide residual groups and thesupport material 40.

Upon immobilizing the fluorescence sensor substance on a surface of asupport material made of an inorganic material or organic material, thesupport material and the fluorescence sensor substance can be chemicallybonded together with a crosslinking agent or the like. Examples of sucha crosslinking agent include silane coupling agents represented by thefollowing formula (10):(R′″O)3-Si-E  Formula (10)

In the formula (10), R′″O represents a C1-C5 alkoxy group such asmethoxy, ethoxy or propoxy, with methoxy or ethoxy being preferred. Aninorganic material can be chemically bonded with such alkoxy groups. Eis a functional group capable of being chemically bonded with an organicmaterial, such as vinyl, epoxy, amino, mercapto, acryl, methacryl,(meth)acryloyl, or a derivative thereof. Illustrative silane couplingagents suitable for use in the present invention includevinylmethoxysilane, 3-glycidoxypropyltrimethoxysilane,N-2-(aminoethyl)-3-aminopropylethoxysilaine,3-mercaptopropyltrimethoxysilane, 3-acryloxypropyltrimethoxysilane,3-methacryloxypropyltrimethoxysilane, and3-methacryloxypropyltriethoxysilane.

Among the above-described silane coupling agents, those containing as Ea substituent group that contains a polymerizable double bond, such asacryl, methacryl or (meth)acryloyl, are also suited for the reasons tobe mentioned below. If such a silane coupling agent is appliedbeforehand on the surface of an inorganic material such as glass ormetal, the fluorescent monomer compound, which contains phenylboronicacid residual groups and acrylamide residual groups, and thepolymerizable monomer, which contains (meth)acrylamide residual group,can be directly immobilized while copolymerizing them together.

To immobilize the fluorescence sensor substance on a surface of anorganic material such as a plastic film, on the other hand, substituentgroups containing reactive groups can be introduced into the plasticfilm to bond them with the fluorescence sensor substance. As a processfor introducing such reactive groups, there is, for example, a graftpolymerization process of glycidyl (meth)acrylate by plasmas, electronrays, radiations or the like as disclosed in Japanese Patent raid-openNo. Hei 5-245198.

To further facilitate the bonding of the fluorescence sensor substanceto an inorganic material or organic material treated with such a silanecoupling agent or to a plastic film in which reactive groups have beenintroduced, a monomer with one or more reactive substituent groupscontained therein may be used beforehand upon synthesis of thefluorescence sensor substance, or subsequent to the synthesis of thefluorescence sensor substance, reactive groups may be introduced. Assuch a monomer, those described above in connection with the florescencesensor substance are each usable. Examples of such reactive groupsinclude amino, carboxyl, hydroxyl, halogenated carboxyl, sulfonyl,thiol, isocyanate, isothiocyanate, and epoxy groups. It is to be notedthat the bonding between the reactive groups and the fluorescence sensorsubstance on the inorganic or organic material treated with the silanecoupling agent can be effected in the presence or absence of suitablesolvent, catalyst and condensing agent.

It is preferred to include a fluorescence detector in the implantable,saccharide-measuring sensor according to the present invention. Moredesirably, such sensor and fluorescence detector can be arrangedtogether with a light source within an adequate housing.

Upon using the fluorescence sensor substance and detector layeraccording to the present invention in the implantable,saccharide-measuring sensor, it is preferred to laminate the opticalisolation layer 20 on the detector layer 10 as illustrated in FIG. 4.When the optical isolation layer 20 is arranged on the side of an outersurface of the sensor, the optical isolation layer 20 can avoid anycontact of the fluorescence sensor substance, which is contained in thedetector layer 10, with radicals, oxidizing substances, reducingsubstances and the like as components of a body fluid, and can protectthe fluorescence sensor substance from deteriorations by such body fluidcomponents. Further, the lamination of the optical isolation layer 20makes it possible to avoid a reduction in detection ability which wouldotherwise take place due to reflection and scattering of excitationlight emitted from the light source 50. Furthermore, even when abiosubstance other than the saccharide is excited by excitation lightfrom the light source, the arrangement of the optical isolation layer 20can shield any light originated from the outside other than theexcitation light from the light source 50, and can also eliminateeffects of any color substances or fluorescent substances in the body.

The optical isolation layer 20 provided with such functions as describedabove is formed of a support material for the optical isolation layerand an opaque material. As the support material for the opticalisolation layer, it is possible to choose a macromolecular materialwhich may be crosslinked or chemically modified. Examples of themacromolecular material include dextran, poly(meth)acrylamides,poly(meth)acrylates, polyethylene glycol, polyvinyl alcohol, polyamides,polyurethanes, their mixtures, and their copolymers. The supportmaterial for the optical isolation layer may be modified with vitamin E,a polyphenol, a metal chelate or the like, or may carry such a compoundthereon. Usable examples of the opaque material include carbon black,fullerene, carbon nanotubes, and iron oxide.

The detector layer 10 and the optical isolation layer 20 can belaminated together via chemical bonds such as covalent bonds, ionicbonds or hydrophobic bonds. When the optical isolation layer usesdextran as its support material and carbon black as its opaque material,for example, dextran is dissolved in a solvent, followed by the additionof carbon black. The resulting mixture is rendered uniform byultrasonication, to which an aqueous solution of an alkali and ethyleneglycol diglycidyl ether are added further. The thus-prepared solution isthen evenly sprayed by a sprayer onto the detector layer, and thedetector layer with the solution sprayed thereon is then heated anddried to laminate an optical isolation layer on the detector layer.

An external view of an implantable, saccharide-measuring sensoraccording to the present invention is shown as a perspective view inFIG. 5. The implantable, saccharide-measuring sensor has a housing 110for keeping liquid-tight the inside of the sensor, a window 120 forexposing only the optical isolation layer or the detector layer, and anantenna portion 130 for performing communications with a system arrangedoutside the body.

The internal structure of the implantable, saccharide-measuring sensoris depicted in FIG. 6. The optical isolation layer 20 or the detectorlayer 10 is arranged such that the window 120 is closed to keep theinside liquid-tight. Also mounted are the light source 50 for emittingexcitation light, an optical waveguide path 170 for guiding light fromthe light source 50 to the detector layer 10, the fluorescence detector60 for detecting fluorescence from the detector layer 10, an integratedcircuit 140 for processing signal data from the fluorescence detector60, and a battery 150 as an internal power supply. On the antennaportion 130, an antenna coil 160 is arranged. It is, however, to benoted that FIG. 5 and FIG. 6 are concept views, and implementations ofthe present invention shall not be limited to these figures. The size,shape and arrangement of each component can be determined freely asneeded.

The use of the implantable, saccharide-measuring sensor makes itpossible to avoid cumbersomeness and the occurrence of time lags in acontinuous blood-sugar measurement when a diabetic controls the bloodsugar level by himself or herself. In addition, the use of theimplantable, saccharide-measuring sensor also allows a non-diabeticperson to simply and easily perform blood sugar level measurements forhealth care.

The present invention will hereinafter be described specifically basedon examples. It should, however, be borne in mind that the followingexamples by no means limit the present invention.

EXAMPLE 1 Synthesis of9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid-1-(6-acrylamido-n-hexyl)amide (hereinafter referred to as “F-AAm”)A) Synthesis of methyl 9,10-bis(bromomethyl)anthracene-2-carboxylate

Methyl 9,10-dimethylanthracene-2-carboxylate (360 mg),N-bromosuccinimide (540 mg) and benzoyl peroxide (5 mg) were added to amixture of chloroform (4 mL) and carbon tetrachloride (10 mL), followedby heating under reflux for 2 hours. Subsequent to the removal of thesolvent, the residue was extracted with methanol to afford the targetcompound (430 mg).

B) Synthesis of methyl 9,10-bis(aminomethyl)anthracene-2-carboxylate

Methyl 9,10-bis(bromomethyl)anthracene-2-carboxylate (400 mg) obtainedin the above procedure A) was dissolved in chloroform (60 mL), a 2 Msolution of methylamine in methanol (8 mL) was added, and the resultingmixture was stirred at room temperature for 4 hours. Subsequent to theremoval of the solvent, the reaction product was purified on a silicagel column with methanol/chloroform as an eluent to afford the targetcompound (235 mg).

C) Synthesis of9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid

Methyl 9,10-bis(aminomethyl)anthracene-2-carboxylate (200 mg) obtainedin the above procedure (B), 2-(2-bromomethylphenyl)-1,3-dioxaborinane(700 mg) and N,N-diisopropylethylamine (0.35 mL) were dissolved indimethylformamide (3 mL), followed by stirring at room temperature for16 hours. Subsequent to removal of the solvent, the reaction product waspurified on a silica gel column with methanol/chloroform as an eluent toafford the methyl ester (194 mg) of the target compound. The methylester was dissolved in methanol (5 mL), and 4 N sodium hydroxide (1 mL)was then added, followed by stirring at room temperature for 10 hours.The reaction mixture was neutralized with hydrochloric acid, and theinorganic salt was removed by gel filtration to afford the targetcompound (180 mg). The melting point of the reaction product was 121°C., and in DMSO-d6, it gave the following 1H-NMR data (δ, ppm): 2.15ppm(d, 6H, N—CH3), 4.10 ppm(m, 4H, N—CH2-benzene), 4.45 ppm(m, 4H,N—CH2-anthracene), 7.55-8.90 ppm(m, 15H,aromatic).

D) Synthesis of F-AAm

In dimethylformamide (5 mL) with N,N-diisopropyl ethylamine (50 mg)contained therein,9,10-Bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid (70 mg) obtained in the above procedure C),1-acrylamido-6-aminohexane (22 mg) and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (50 mg) were dissolved,followed by stirring at 60° C. for 18 hours. The reaction mixture wasdissolved in chloroform (50 mL). The thus-prepared solution was washedthree times with distilled water and once with saturated salinesolution. The chloroform layer was dried with anhydrous sodium sulfate,and was then distilled to dryness under reduced pressure to afford thetarget compound (85 mg).

EXAMPLE 2 Synthesis of9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid-(terminal acrylamido-PEG3400)amide

9,10-Bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid (10 mg) obtained in the above procedure C) of Example 1,amino-PEG3400-acrylamide (product of Nektar Corp.; 50 mg) and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (22 mg) were dissolvedin 100 mM phosphate buffer (pH 6.0; 3 mL), followed by stirring at 60°C. for 24 hours. The reaction mixture was subjected to gel filtration,and a fluorescent polymer fraction was collected to afford the targetcompound (36 mg).

EXAMPLES 3 TO 9

Prepared were a dimethylsulfoxide (hereinafter referred to as “DMSO”)solution having an F-AAm concentration of 10 wt %, a 80 wt % DMSO-watermixed solution having an acrylamide (hereinafter referred to as “AAm”)concentration of 30 wt %, a 50 wt % DMSO-water mixed solution having asodium persulfate (hereinafter referred to as “SPS”) concentration of 3wt %, and a DMSO solution having an N,N,N′,N′-tetramethylethylenediamine(hereinafter abbreviated as “TEMED”) concentration of 2 wt %. Usingthose reagent solutions, DMSO and water, reaction solutions wereprepared to give their final concentrations and compositions as shown inTable 1. The thus-prepared reaction solutions were subjected topolymerization at room temperature for 2 hours to obtain copolymers ofF-AAm and AAm. They will be referred to as Examples 3-9, respectively.The resultant copolymers were separately caused to precipitate fromacetone, dissolved in water, and reprecipitated from acetone. Thisprocedure was repeated twice to conduct purification. Subsequent to thepurification, the resulting fluorescence sensor substances were dried invacuo.

TABLE 1 F-AAm AAm SPS TEMED DMSO Total F-AAm/AAm (wt %) (wt %) (wt %)(wt %) (wt %) amount (mL) Charged molar ratio Example 3 1.46 2.1 0.150.04 80 2.0 1/14  Example 4 0.43 1/50  Example 5 0.15 1/144 Example 60.10 1/215 Example 7 0.050 1/430 Example 8 0.015  1/1435 Example 90.0075  1/2870

EXAMPLE 10 Production of 9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid-(terminal acrylamido-PEG3400)amide/AAm copolymer

Prepared were an aqueous solution with9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid-(terminal acrylamido-PEG3400)amide contained at 10 wt %, an aqueoussolution with AAm contained at 30 wt %, a water mixed solution with SPScontained at 3 wt %, and an aqueous solution with TEMED contained at 2wt %. With those reagent solutions, and water, a reaction solution wasprepared containing a fluorescent monomer compound, which was the sameas that synthesized in Example 2, at 0.6 wt %, AAm at 4.5 wt %, SPS at0.3 wt % and TEMED at 0.08 wt %, all as final concentrations (1/427 interms of the charged molar ratio of the fluorescent monomer compound toAAm). The reaction solution was subjected to polymerization at roomtemperature for 8 hours to afford a copolymer. The copolymer was causedto separately precipitate from acetone, dissolved in water, andreprecipitated from acetone. This procedure was repeated ten times toconduct purification. Subsequent to the purification, the resultingfluorescence sensor substance was dried in vacuo.

EXAMPLE 11

The copolymers obtained in Examples 3 to 10 were each dissolved in a 1/2(v/v) solution of methanol and phosphate buffer to give a concentrationof 0.05 mg/mL. Using a spectrophotometer, the absorbance at 265 nm wasmeasured. Concerning an acrylamide homopolymer (molecular weight:150,000), the absorbance was also measured likewise. This absorbance wassubtracted, as a BLANK value, from the absorbance values of thecopolymers of the respective examples.

Based on calibration curves prepared beforehand for the respectivefluorescent monomer compounds, the fluorescent monomer compound/AAmratios of the individual fluorescence sensor substances were determined.The results are shown in Table 2.

It is appreciated from Table 2 that in Example 3 to 9, the absorbanceincreased in proportion to the content of charged F-AAM and hence, F-AAmwas incorporated at a fixed rate as a component as the respectivefluorescence sensor substances.

TABLE 2 Fluorescent monomer Fluorescent monomer compound/AAmBlank-corrected compound/AAm Charged molar ratio Absorbance absorbanceMeasured molar ratio Example 3 1/14  0.725 0.698 1/10  Example 4 1/50 0.231 0.204 1/59  Example 5 1/144 0.113 0.086 1/185 Example 6 1/2150.076 0.049 1/282 Example 7 1/430 0.052 0.025 1/572 Example 8  1/14350.035 0.008  1/1932 Example 9  1/2870 0.031 0.004  1/3874 Example 101/427 0.032 0.005  1/3534Calibration curve formula for fluorescent monomer compounds: y=20.158x(r=1, x: F-AAm concentration[μmol/mL])

EXAMPLE 12

To study the responses of the fluorescent monomer compounds in thecopolymers, which had been synthesized in Examples 3 to 10, influorescence intensity to a glucose concentration by making equal theconcentrations of the copolymers, the copolymers were each dissolved inphosphate buffer of pH 7.0 to give a concentration such that theabsorbance at 265 nm would become 0.05. As shown in FIG. 7, among therelative fluorescence intensitys (Ex=405 nm, Em=442 nm) of theindividual fluorescence sensor substance solutions at a glucoseconcentration of 500 mg/dL, the relative fluorescence intensity of thefluorescence sensor substance (F-AAm/AAm=1/14) of Example 3 was lowercompared with those of the fluorescence sensor substances of the sameF-AAm/AAm copolymer system. On the other hand, the fluorescence sensorsubstance of Example 10, which had been synthesized using thefluorescence monomer compound with the extended “Y” moiety containedtherein, showed very high relative fluorescence intensity.

EXAMPLE 13

The responses in fluorescence intensity to the glucose concentration of500 mg/dL were studied in a similar manner as in Example 12 except thatthe concentrations of the copolymers synthesized in Examples 3 to 9 wereadjusted equally at 10 μg/mL. The results are shown in FIG. 8.

EXAMPLE 14

A solution was prepared by dissolving and mixing AAm,N,N′-methylenebisacrylamide (hereinafter referred to as “BIS”), SPS,TEMED and F-AAm reagent solutions in an 80 wt % DMSO-water solution togive the following final concentrations: AAm: 15 wt %, BIS: 0.15 wt %,SPS: 0.3 wt %, TEMED: 0.08 wt %, and F-AAm: 0.50 wt % (F-AAm/AAm=1/300in terms of the charged molar ratio).

A glass plate, which had been subjected beforehand to surface treatmentwith a silane coupling agent, and an untreated glass plate were arrangedwith a space left therebetween. The solution was poured into the space,followed by polymerization at room temperature for 2 hours under anitrogen atmosphere. Subsequent to the completion of the polymerization,the glass plates were dipped in pure water, and only the untreated glassplate was separated to obtain a gel sheet formed of an F-AAm/AAmcopolymer immobilized on the glass-made support material. Thethus-obtained gel sheet was alternately dipped in methanol and 50 mMphosphate buffer (pH=7.0) thrice for 5 minutes per dip, and was thenimmersed and washed for 10 hours or longer in phosphate buffer to obtaina detector layer.

EXAMPLE 15

In dimethylformamide (30 mL) with N,N-diisopropylethylamine (0.2 mL)contained therein,9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid (350 mg) synthesized in the procedure C) of Example 1, methyl6-aminohexanoate (200 mg) and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (220 mg) were dissolved,followed by stirring at room temperature for 4 hours. The reactionmixture was dissolved in chloroform (100 mL). The thus-prepared solutionwas washed three times with distilled water and once with saturatedsaline solution. The chloroform layer was dried with anhydrous sodiumsulfate, and was then distilled to dryness under reduced pressure toafford its methyl ester (360 mg) as an intermediate. The intermediatewas dissolved in methanol (10 mL), and subsequent to the addition of a 4N aqueous solution of sodium hydroxide (1 mL), they were reacted at roomtemperature for 15 hours. The reaction mixture was loaded on an ionexchange column to remove the alkali, and was then concentrated todryness to afford9,10-bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid-1-(6′-carboxylic acid-n-hexyl)amide (310 mg).

EXAMPLE 16

An aqueous solution of perchloric acid (effective chlorineconcentration: 5 wt %, 8 g) and a 6 N aqueous solution of sodiumhydroxide (30 mL) were taken in a shallow square stainless-steelcontainer of 10 cm×10 cm, and then chilled to 0° C. A polyacrylamidemembrane, which had been cut into a square of 10 cm×10 cm, was gentlyplaced in the container, and was reacted at 0° C. for 2 hours. Thereaction mixture was removed, and the resulting membrane was gentlywashed 4 times with distilled water (40 mL, each) and twice withdimethylformamide (20 mL, each) to obtain an activated polyacrylamidemembrane.

9,10-Bis[[N-methyl-N-(ortho-boronobenzyl)amino]methyl]anthracene-2-carboxylicacid-1-(6′-carboxylic acid-n-hexyl)amide (20 mg), the fluorescentmonomer compound synthesized in Example 15,1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (12 mg) and1-hydroxybenzotriazole (8 mg) were dissolved in dimethylformamide (10mL). The resulting solution was poured into a shallow squarestainless-steel container of 10 cm×10 cm, and the activatedpolyacrylamide membrane prepared in the above-described reaction wassoaked. After the membrane was reacted at room temperature for 17 hours,it was washed thrice with dimethylformamide (20 mL, each), twice with0.01 N hydrochloric acid (40 mL, each) and thrice with distilled water(40 mL, each), and further, was immersed and washed for 10 hours orlonger in 50 mM phosphate buffer (pH=7.0) to obtain a detector layerwith the fluorescent monomer compound immobilized on the polyacrylamidemembrane.

Dextran (7 g) was dissolved at 50° C. under stirring in distilled water(175 mL), and subsequent to the addition of carbon black (5 g), theresulting mixture was subjected to ultrasonication until the carbonblack was evenly dispersed. To the mixture, a 50 wt % aqueous solutionof sodium hydroxide (3.5 mL) and ethylene glycol diglycidyl ether (6.5g) were added, followed by stirring at 45° C. for 30 minutes. Distilledwater (230 mL) was then added, and the resulting mixed solution wasplaced in a sprayer. The detector layer prepared in the preceding stepwas held in place on a flat glass plate, the mixed solution was sprayedevenly, and then, drying was conducted for 30 minutes in an ovencontrolled at 45° C. to obtain the detector layer with an opticalisolation layer laminated thereon.

COMPARATIVE EXAMPLE 1 synthesis and polymerization of9,10-bis(methylene)[N-(acryloylaminohexyl)-N-(ortho-boronobenzyl)methylene]anthracene(hereinafter referred to as “F-AAm-2”) A′) Synthesis of9,10-bis[6′-(t-butoxycarbonylamino)hexylaminomethyl]anthracene

9,10-Bis(chloromethyl)anthracene (500 mg), N-BOC-hexyldiamine (1.3 g)and diisopropylethylamine (1.25 mL) were dissolved in dimethyl sulfoxide(10 mL), followed by reaction under stirring at 60° C. for 6 hours. Thereaction mixture was diluted with chloroform (60 mL), and washed thricewith water (100 mL, each) and once with saturated saline solution (100mL). The organic layer was dried over anhydrous sodium sulfate. Afterthe desiccant was filtered off, the organic layer was concentrated, andthe concentrate was purified by chromatography on a silica gel columnwith chloroform/methanol as an eluent to afford the target compound (56mg).

B′) Synthesis of9,10-bis[[N-6′-(t-butoxycarbonylamino)hexyl-N-[2-(5,5-dimethylborinan-2-yl)benzyl]amino]methyl]anthracene

The product (50 mg) obtained in the above procedure A′),2-(2-bromomethylphenyl)-1,3-dioxaborinane (170 mg) andN,N-diisopropylethylamine (0.1 mL) were dissolved in dimethylformamide(1 mL), followed by stirring at 25° C. for 5 hours. Subsequent to theremoval of the solvent, the reaction product was purified on a silicagel column with methanol/chloroform as an eluent to afford the targetcompound (35 mg).

C′) Synthesis of9,10-bis[[N-6′-aminohexyl-N-(ortho-boronobenzyl)amino]methyl]anthracene

The product (35 mg) obtained in the above procedure B′) was dissolved inmethanol (1 mL). 4 N hydrochloric acid (0.5 mL) was added, and thethus-obtained mixture was stirred at 25° C. for 10 hours. Subsequent toevaporation to dryness, the inorganic salt was removed by gel filtrationto afford the target compound (26 mg).

D′) Synthesis of F-AAm-2

The product (25 mg) obtained in the above procedure C′) was dissolved inDMF (0.5 mL). Under a nitrogen atmosphere, N,N-diisopropylethylamine (20mg) and acryloyl chloride (12 mg) were added at −10° C., followed byreaction under stirring for 30 minutes. The reaction mixture was pouredinto iced water, extracted with chloroform, and washed with saturatedsaline solution. The chloroform layer was dried, and was then evaporatedto dryness to afford the target product represented by thebelow-described formula (11) (26 mg) as a comparative compound.

E′) Polymerization

A solution was prepared by dissolving and mixing AAm monomer, BIS, SPS,TEMED and F-AAm-2 represented by the formula (11) in an 80 wt %DMSO-water solution to give the following final concentrations: AAmmonomer: 15 wt %, BIS: 0.15 wt %, SPS: 0.3 wt %, TEMED: 0.08 wt %, andF-AAm-2: 0.50 wt % (F-AAm-2/AAm=1/300 in terms of the charged molarratio).

A glass plate, which had been subjected beforehand to surface treatmentwith a silane coupling agent, and an untreated glass plate were arrangedwith a space left therebetween. The solution was poured into the space,followed by polymerization at room temperature for 2 hours under anitrogen atmosphere. Subsequent to the completion of the polymerization,the glass plates were dipped in pure water, and only the untreated glassplate was separated to obtain a gel sheet formed of an F-AAm-2/AAmcopolymer immobilized on the glass-made support material. Thethus-obtained gel sheet was alternately dipped in methanol and 50 mMphosphate buffer (pH=7.0) thrice for 5 minutes per dip, and was thenimmersed and washed for 10 hours or longer in phosphate buffer to obtaina detector layer preform. An optical isolation layer was laminated onthe detector layer preform in a similar manner as in Example 16 toprovide a detector layer.

EXAMPLE 17 Synthesis of9,10-bis(methylene)[[N-(ortho-boronobenzyl)methylene]-N-[(acryloylpolyoxyethylene)carbonylamino]-n-hexamethylene]-2-acetylanthracene(hereinafter referred to as “F-PEG-AAm-1”) A) Synthesis of9,10-bis(bromomethyl)-2-acetylanthracene

9,10-Dimethyl-2-acetylanthracene (600 mg), N-bromosuccinimide (800 mg)and benzoyl peroxide (5 mg) were added to a mixture of chloroform (6 mL)and carbon tetrachloride (20 mL), followed by heating under reflux at80° C. for 2 hours. After the solvent was eliminated, the residue wasextracted with methanol to afford the target compound (780 mg).

B) Synthesis of9,10-bis[6′-(t-butoxycarbonylamino)hexylaminomethyl]-2-acetylanthracene

The product (500 mg) obtained in the above procedure A),N-BOC-hexyldiamine (1.125 g) and diisopropylethylamine (1.25 mL) weredissolved in dimethylformamide (10 mL), followed by reaction understirring at 45° C. for 1 hour. The reaction mixture was diluted withchloroform (60 mL), and then washed thrice with water (100 mL, each) andonce with saturated saline solution (100 mL). The organic layer wasdried over anhydrous sodium sulfate. After the desiccant was filteredoff, the organic layer was concentrated, and the concentrate waspurified by chromatography on a silica gel column withchloroform/methanol as an eluent to afford the target compound (367 mg).

C) Synthesis of9,10-bis[[N-6′-(t-butoxycarbonylamino)hexyl-N-[2-(5,5-dimethylborinan-2-yl)benzyl]amino]methyl]-2-acetylanthracene

The product (200 mg) obtained in the above procedure B),2-(2-bromomethylphenyl)-1,3-dioxaborinane (700 mg) andN,N-diisopropylethylamine (0.35 mL) were dissolved in dimethylformamide(3 mL), followed by stirring at 25° C. for 16 hours. Subsequent to theremoval of the solvent, the reaction product was purified on a silicagel column with methanol/chloroform as an eluent to afford the targetcompound (194 mg).

D) Synthesis of9,10-bis[[N-6′-aminohexyl-N-(ortho-boronobenzyl)amino]methyl]-2-acetylanthracene

The product (100 mg) obtained in the above procedure C) was dissolved inmethanol (2 mL). 4 N hydrochloric acid (2 mL) was added, and thethus-obtained mixture was stirred at 25° C. for 10 hours. Subsequent toevaporation to dryness, the inorganic salt was removed by gel filtrationto afford the target compound (95 mg).

E) Synthesis of F-PEG-AAm-1

The product (160 mg) obtained in the above procedure D) was dissolved indimethylformamide (0.5 mL). The solution was added to a solution ofacryloyl-(polyethylene glycol)-N-hydroxysuccinimide ester (PEG molecularweight: 3,400; 1.22 g) in 100 mM phosphate buffer (pH=8.0; 10 mL),followed by stirring at 25° C. for 20 hours. The reaction mixture wassubjected to gel filtration to collect a fluorescent macromoleculefraction. Subsequent to lyophilization, the target compound was obtained(1.2 g). The above procedures A) to E) are shown in the synthesis schemeof FIG. 1. F-PEG-AAm-1 was dissolved at 100 mM concentration in waterunder the conditions of 25° C. temperature and pH 7.0 without thepresence of any organic solvent or solubilizer. The 1H-NMR data of thetarget compound in deuterochloroform were as follows (δ, ppm):1.30-1.65(m, C—CH2-C), 2.90(s, Ac), 2.78(m, —C(—C)N—CH2-C), 3.25(m,CH2-NH—COO), 3.50-3.80(s, PEG), 5.80(d, COCH═C), 6.17(m, C═CH2),7.20-8.20(m, aromatic). No hydrogen signal was confirmed to overlap apeak ascribable to PEG residual groups.

EXAMPLE 18

A solution was prepared by dissolving and mixing acrylamide, F-PEG-AAm-1synthesized in Example 17, methylene-bisacrylamide, sodium persulfateand N,N,N′,N′-tetramethylethylenediamine in pure water to give thefollowing final concentrations: acrylamide: 15 wt %, F-PEG-AAm-1: 10 wt%, methylene-bisacrylamide: 0.3 wt %, sodium persulfate: 0.18 wt %, andN,N,N′,N′-tetramethylethylenediamine: 0.36 wt %. A glass plate, whichhad been subjected beforehand to surface treatment with a silanecoupling agent, and an untreated glass plate were arranged with a spaceleft therebetween. The solution was poured into the space, followed bypolymerization at 25° C. for 8 hours under a nitrogen atmosphere.Subsequent to the completion of the polymerization, the glass plateswere dipped in pure water, and only the untreated glass plate wasseparated to obtain a gel sheet formed of an acrylamide/F-PEG-AAm-1(molar ratio: 160/1) copolymer immobilized on the glass-made supportmaterial.

A solution was prepared by dissolving or mixing acrylamide,methylene-bisacrylamide, sodium persulfate,N,N,N′,N′-tetramethylethylenediamine and carbon black in pure water togive the following final concentrations: acrylamide: 20 wt %,methylene-bisacrylamide: 1 wt %, sodium persulfate: 0.18 wt %,N,N,N′,N′-tetramethylethylenediamine: 0.36 wt %, and carbon black: 5 wt%. The solution was applied to a surface of the gel sheet, followed bypolymerization at 25° C. for 24 hours under a nitrogen atmosphere. Theresulting gel sheet was dipped and washed ten times in total for 30minutes per dip in 50 mM phosphate buffer (pH 7.0) having a glucoseconcentration of 500 mg/dL. Subsequently, the gel sheet was dipped andwashed 4 times in total for 30 minutes per dip in 50 mM phosphate buffer(pH 7.0) such that glucose was washed off to obtain a detector layer.

EXAMPLE 19 synthesis of methyl9,10-bis(methylene)[[N-(ortho-boronobenzyl)]-N-[(acryloylpolyoxyethylene)carbonylamino]-n-hexamethylene]-anthracene-2-carboxylate(hereinafter referred to as “F-PEG-AAm-2”) a) Synthesis of9,10-dimethylanthracene-2-carboxylic acid

9,10-Dimethyl-2-acetylanthracene (1.2 g) was dissolved in dioxane (24mL). An aqueous solution of sodium perchlorate (10 mL; effectivechlorine concentration: 10 wt %) was then added, followed by reactionunder stirring at 85° C. for 8 hours. The reaction mixture was chilled,and then rendered acidic with dilute hydrochloric acid. The resultingprecipitate was collected by filtration, washed with a small amount ofwater, and then dried in vacuo to afford the target compound (1.16 g).

b) Synthesis of methyl 9,10-dimethylanthracene-2-carboxylate

The product (1 g) obtained in the above procedure a) was dissolved in 5wt % hydrochloric acid-methanol, followed by heating under reflux for 20hours. The reaction mixture was concentrated, and water (30 mL) wasadded. The resulting mixture was extracted with chloroform (100 mL). Thechloroform layer was washed with an aqueous solution of sodiumbihydrogencarbonate and saturated saline solution, and was then driedover anhydrous sodium sulfate. The chloroform layer was evaporated todryness to afford the target compound (915 mg).

c) Synthesis of methyl 9,10-bis(bromomethylene)anthracene-2-carboxylate

The product (600 mg) obtained in the above procedure b),N-bromosuccinimide (800 mg) and benzoyl peroxide (5 mg) were added to amixture of chloroform (6 mL) and carbon tetrachloride (20 mL), followedby heating under reflux for 2 hours. Subsequent to the removal of thesolvent, the residue was extracted with methanol to afford the targetcompound (767 mg).

d) Synthesis of methyl9,10-bis[6′-(t-butoxycarbonylamino)hexylaminomethyl]anthracene-2-carboxylate

The product (500 mg) obtained in the above procedure c),N-BOC-hexyldiamine (1.125 g) and diisopropylethylamine (1.25 mL) weredissolved in dimethylformamide (10 mL), followed by reaction understirring at 45° C. for 2 hours. The reaction mixture was diluted withchloroform (60 mL), and then washed thrice with water (100 mL, each) andonce with saturated saline solution (100 mL). The organic layer wasdried over anhydrous sodium sulfate. After the desiccant was filteredoff, the organic layer was concentrated, and the concentrate waspurified by chromatography on a silica gel column withchloroform/methanol as an eluent to afford the target compound (307 mg).

e) Synthesis of methyl9,10-bis[[N-6′-(t-butoxycarbonylamino)hexyl-N-[2-(5,5-dimethylborinan-2-yl)benzyl]amino]methyl]anthracene-2-carboxylate

The product (200 mg) obtained in the above procedure d),2-(2-bromomethylphenyl)-1,3-dioxaborinane (700 mg) andN,N-diisopropylethylamine (0.35 mL) were dissolved in dimethylformamide(3 mL), followed by stirring at 25° C. for 16 hours. Subsequent to theremoval of the solvent, the reaction product was purified on a silicagel column with methanol/chloroform as an eluent to afford the targetcompound (203 mg).

f) Synthesis of methyl9,10-bis(methylene)[[N-(ortho-boronobenzyl)methylene]-N-(aminohexyl)]anthracene-2-carboxylate

The product (100 mg) obtained in the above procedure e) was dissolved inmethanol (3 mL). 8 N hydrochloric acid (0.5 mL) was added, and thethus-obtained mixture was stirred at 25° C. for 2 days. Subsequent toevaporation to dryness, the inorganic salt was removed by gel filtrationto afford the target compound (82 mg).

g) Synthesis of F-PEG-AAm-2

The product (160 mg) obtained in the above procedure f) was dissolved indimethylformamide (0.5 mL). The solution was added to a solution ofacryloyl-(polyethylene glycol)-N-hydroxysuccinimide ester (PEG molecularweight: 3,400; 1.22 g) in 100 mM phosphate buffer (pH=8.0; 10 mL),followed by stirring at 25° C. for 20 hours. The reaction mixture wassubjected to gel filtration to collect a fluorescent macromoleculefraction. Subsequent to lyophilization, the target compound was obtained(1.1 g). The above procedures a) to e) are shown in the synthesis schemeof FIG. 3. F-PEG-AAm-2 was dissolved at 100 mM concentration in waterunder the conditions of 25° C. temperature and pH 7.0 without thepresence of any organic solvent or solubilizer. The 1H-NMR data of thetarget compound in deuterochloroform were as follows (δ, ppm):1.30-1.65(m, C—CH₂—C), 2.78(m, —C(—C)N—CH₂—C), 3.25(m, CH₂—NH—COO),3.50-3.80(s, PEG), 4.10(s, COOCH ₃), 5.80(d, COCH═C), 6.17(m, C═CH₂),7.20-8.20(m, aromatic). No hydrogen signal was confirmed to overlap apeak ascribable to PEG residual groups.

EXAMPLE 20

Using F-PEG-AAm-2 synthesized in Example 19, a gel sheet formed of anacrylamide/F-PEG-AAm-2 (molar ratio: 160/1) immobilized on a glass-madesupport material was obtained in a similar manner as in Example 18.Further, a detector layer was obtained in a similar manner as in Example18.

COMPARATIVE EXAMPLE 2 Synthesis and Polymerization of AAm-2

F-AAm-2 represented by the formula (11) was synthesize in a similarmanner as in the procedures A′) to D′) of Comparative Example 1.

E″) Polymerization

A solution was prepared by dissolving and mixing acrylamide,methylene-bisacrylamide, F-AAm-2, sodium persulfate andN,N,N′,N′-tetramethylethylenediamine in an 80 v/v % aqueous solution ofdimethylsulfoxide to give the following final concentrations:acrylamide: 15 wt %, methylene-bisacrylamide: 0.3 wt %, F-AAm-2: 1.2 wt%, sodium persulfate: 0.18 wt %, andN,N,N′,N′-tetramethylethylenediamine: 0.36 wt %.

A glass plate, which had been subjected beforehand to surface treatmentwith a silane coupling agent, and an untreated glass plate were arrangedwith a space left therebetween. The solution was poured into the space,followed by polymerization at 25° C. for 8 hours under a nitrogenatmosphere. Subsequent to the completion of the polymerization, theglass plates were dipped in pure water, and only the untreated glassplate was separated to obtain a gel sheet formed of anacrylamide/F-AAm-2 (molar ratio: 150/1) copolymer immobilized on theglass-made support material.

A solution was prepared by dissolving or mixing acrylamide,methylene-bisacrylamide, sodium persulfate,N,N,N′,N′-tetramethylethylenediamine and carbon black in pure water togive the following final concentrations: acrylamide: 20 wt %,methylene-bisacrylamide: 1 wt %, sodium persulfate: 0.18 wt %,N,N,N′,N′-tetramethylethylenediamine: 0.36 wt %, and carbon black: 5 wt%. The solution was applied to a surface of the gel sheet, followed bypolymerization at 25° C. for 24 hours under a nitrogen atmosphere. Theresulting gel sheet was dipped and washed ten times in total for 30minutes per dip in 50 mM phosphate buffer (pH 7.0) having a glucoseconcentration of 500 mg/dL. Subsequently, the gel sheet was dipped andwashed 4 times in total for 30 minutes per dip in 50 mM phosphate buffer(pH 7.0) such that glucose was washed off to obtain a detector layer.

EXAMPLE 21

The detector layers obtained in Example 16 and Comparative Example 1were held in place on evaluation devices, respectively. Under phosphatebuffer (pH=7.0), their responses to glucose at varied concentrationswere evaluated in fluorescence intensity. The results are shown in FIG.9.

Each evaluation device was provided with a cell, a bundle of opticalfibers, and a fluorospectrometer. The cell can fixedly accommodate thedetector layer, and permits circulation of a liquid through the insidethereof. The bundle of optical fibers is fixedly secured at one endthereof on a rear side of the cell, and is connected at an opposite endthereof to the fluorospectrometer. A fraction of the bundled opticalfibers is used to feed exciting light to the cell, and the remainingfraction of the bundled optical fibers is used to return fluorescentradiation from the cell.

As clearly envisaged from FIG. 9, it is appreciated that the detectorlayer of Example 16 is superior in the response to glucose compared withthe detector layer of Comparative Example 1.

EXAMPLE 22

The detector layers obtained in Example 18, Example 20 and ComparativeExample 2 were held in place on evaluation devices, respectively. Underphosphate buffer (pH=7.0), their responses to glucose at variedconcentrations were evaluated in fluorescence intensity. The results areshown in FIG. 10. The evaluation devices used were similar to thoseemployed in Example 21.

As clearly envisaged from FIG. 10, it is evident that the detectorlayers of Examples 18 and 20, which had been produced using thewater-soluble monomer, were superior in the response to glucose comparedwith the detector layer of Comparative Example 2 produced using theconventional hydrophobic monomer.

EXAMPLE 23

The detector layers obtained in Examples 16, 18 and 20 and ComparativeExample 2 were held in place on evaluation devices, respectively. Underphosphate buffer (pH=7.0), fluorescent spectra were measured.

The detector layer of Comparative Example 2 showed maximum fluorescencewavelengths at 403 nm and 430 nm when excited at 377 nm. On the otherhand, the detector layer of Example 16 showed a maximum fluorescencewavelength at 450 nm when excited at 400 nm, the detector layer ofExample 18 showed a maximum fluorescence wavelength at 480 nm whenexcited at 400 nm, and the detector layer of Example 20 showed a maximumfluorescence wavelength at 455 nm when excited at 400 nm. Each detectorlayer according to the present invention has a fluorescent wavelengthshifted toward the longer side, has a great difference between itsexcitation wavelength and fluorescence wavelength, is advantageous fromthe standpoint of fluorescence characteristics, and is expected toprovide high measurement accuracy.

1. A fluorescent monomer compound represented by the following formula(1):

wherein: Q, Q′ and D³ may be the same or different, may be combinedtogether into a fused ring, and are each a substituent selected from thegroup consisting of a hydrogen atom, a halogen atom, a hydroxyl group,and substituted or unsubstituted alkyl, acyl, oxyalkyl, carboxyl,carboxylate ester, carboxamido, cyano, nitro, amino and aminoalkylgroups; and D¹, D² and D⁴ each represent a substituent, wherein at leastone of D¹, D² and D⁴ is a substituent group comprising a vinyl group atan end thereof, and wherein the substituent group comprising a vinylgroup at an end thereof enables the fluorescent monomer compound to besoluble in water, and the substituent group comprising a vinyl group atan end thereof is represented by the following formula (2) or formula(3):

wherein: X in formula (2) is a substituent group selected from the groupconsisting of —COO—, —OCO—, —CH₂NR—, —CH₂S—, —CH₂O—, —NR—, —NRCO—,—CONR—, —SO₂NR—, —NRSO₂—, —O—, —S—, —SS—, —NRCOO—, —OCONR— and —CO—; andX in formula (3) represents a C1 to C30 alkylene group comprising atleast one substituent group selected from the group consisting of —COO—,—OCO—, —CH₂NR—, —NR—, —NRCO—, —CONR—, —SO₂NR—, —NRSO₂—, —O—, —S—, —SS—,—NRCOO—, —OCONR— and —CO—, in which R represents a hydrogen atom or asubstituted or unsubstituted alkyl group, Y is a substituted orunsubstituted, divalent, organic residual group containing a structurerepresented by the following formula (4) or formula (5):

wherein n is from 2 to 5, j is from 1 to 5, m is from 1 to 200, and Y′and Y″ may be the same or different and are each a hydrogen atom or analkyl group, and Z represents —O— or —NR″—, and R″ represents a hydrogenatom or a substituted or unsubstituted alkyl group.
 2. A fluorescentmonomer compound represented by the following formula (1):

wherein: Q and Q′ may be the same or different, may be combined togetherinto a fused ring, and are each a substituent selected from the groupconsisting of a hydrogen atom, a halogen atom, a hydroxyl group, andsubstituted or unsubstituted alkyl, acyl, oxyalkyl, carboxyl,carboxylate ester, carboxamido, cyano, nitro, amino and aminoalkylgroups; and in D¹, D², D³ and D⁴, (i) D¹ and D² may be the same ordifferent and are each a substituted or unsubstituted alkyl group, D³ isa hydrogen atom, and D⁴ is a substituent group represented by thefollowing formula (2), or (ii) D¹ and D² may be the same or differentand are each a substituent group represented by the following formula(3), and D³ and D⁴ may be the same or different, may be combinedtogether into a fused ring, and are each a substituent selected from thegroup consisting of a hydrogen atom, a halogen atom, a hydroxyl group,and substituted or unsubstituted alkyl, acyl, oxyalkyl, carboxyl,carboxylate ester, carboxamido, cyano, nitro, amino and aminoalkylgroups:

wherein: X, in a case of the definition (i), is a substituent groupselected from the group consisting of —COO—, —OCO—, —CH₂NR—, —CH₂S—,—CH₂O—, —NR—, —NRCO—, —CONR—, —SO₂NR—, —NRSO₂—, —O—, —S—, —SS—, —NRCOO—,—OCONR— and —CO—, and in a case of the definition (ii), represents a C1to C30 alkylene group comprising at least one substituent group selectedfrom the group consisting of —COO—, —OCO—, —CH₂NR—, —NR—, —NRCO—,—CONR—, —SO₂NR—, —NRSO₂—, —O—, —S—, —SS—, —NRCOO—, —OCONR— and —CO—, inwhich R represents a hydrogen atom or a substituted or unsubstitutedalkyl group, Y is a substituted or unsubstituted, divalent, organicresidual group containing a structure represented by the followingformula (4) or formula (5):

wherein n is from 2 to 5, j is from 1 to 5, m is from 1 to 200, and Y′and Y″ may be the same or different and are each a hydrogen atom or analkyl group, and Z represents —O— or —NR″—, and R″ represents a hydrogenatom or a substituted or unsubstituted alkyl group.
 3. The fluorescentmonomer compound according to claim 2, wherein said substituent grouprepresented by the formula (2) or said substituent group represented bythe formula (3) enables the fluorescent monomer compound to be solublein water.
 4. The fluorescent monomer compound according to claim 2,wherein D₁, D₂, D₃ and D₄ have the meanings as defined under thedefinition (i), and Y is a substituted or unsubstituted, linear,divalent, organic residual group having a number of atoms of from 3 to500.